Macrophage Activation and Differentiation Signals Regulate Schlafen-4 Gene Expression: Evidence for Schlafen-4 as a Modulator of Myelopoiesis

Background: The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Potent T cell response to a class I-binding 13-mer viral epitope and the influence of HLA micropolymorphism in controlling epitope length

The BZLF1 antigen of Epstein-Barr virus includes three overlapping sequences of different lengths that conform to the binding motif of human leukocyte antigen (HLA) B*3501. These 9-mer ((56)LPOGQLTAy(64)), 11-mer ((54)EPLPQGQLTAy(64)), and 13-mer ((52)LPEPLPQGQLTAY(64)) peptides all bound well to B*3501; however, the CTL response in individuals expressing this HILA allele was directed strongly and exclusively towards the 11-mer peptide. In contrast, EBV-exposed donors expressing HLA B*3503 showed no significant CTL response to these peptides because the single amino acid difference between B*3501 and B*3503 within the F pocket inhibited HLA binding by these peptides. The extraordinarily long 13-mer peptide was the target for the CTL response in individuals expressing B*3508, which differs from B*3501 at a single position within the D pocket (B*3501, 156 Leucine; B*3508, 156 Arginine). This minor difference was shown to enhance binding of the 13-mer peptide, presumably through a stabilizing interaction between the negatively charged glutamate at position 3 of the peptide and the positively charged arginine at HLA position 156. The 13-mer epitope defined in this study represents the longest class I-binding viral epitope identified to date as a minimal determinant. Furthermore, the potency of the response indicates that peptides of this length do not present a major structural barrier to CTL recognition

Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice

We generated double-transgenic mice carrying cointegrated tissue-specific Gal4 and Gal4 reporter transgenes to direct transgene overexpression in the mononuclear phagocyte system (MPS). A modified promoter of the Csf1r (c-fms) gene, containing a deletion of the trophoblast-specific promoter, was used to drive the expression of Gal4VP16 transcriptional activator specifically in macrophages. This module was cointegrated with a fluorescent reporter, enhanced cyan fluorescent protein (ECFP), driven by a Gal4-dependent promoter. ECFP fluorescence was first detected in forming blood islands of the yolk sac at 8 dpc, then in macrophages in the yolk sac and the embryo proper. In adult mice ECFP was detected primarily in monocytes, tissue macrophages, microglia, and dendritic cells, including Langerhans cells of the skin. Crossing of these mice to transgenics containing tagged protein under control of a Gal4-dependent promoter directed expression of that protein in mononuclear phagocytes of double-transgenic animals. The new mouse line provides a useful tool for overexpression of transgenes in cells of the myeloid lineage, while simultaneously labeling them by ECFP expression

Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

<div><p>Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.</p> </div

p115 and Sec16A associate with TRAF3 following cytosolic RNA and DNA sensor activation.

<p>(<b>A</b>) Whole-cell lysates (HeLa cells) were prepared and subjected to immunoprecipitation assays using TRAF3 (H-20) or isotype control antibodies followed by immunoblotting for the presence of p115, Sec16A, and TRAF3. (<b>B</b>) HeLa cells were treated as indicated for different periods of time. Whole-cell lysates were prepared and subjected to immunoprecipitation assays using TRAF3 (H-20) antibody followed by immunoblotting for the presence of p115, Sec16A, TBK1 and TRAF3. The Native-PAGE assay was conducted on the same cellular extracts to demonstrate the dimerization and activation of IRF-3 upon indicated treatments. One of three independent experiments with similar results is shown.</p

Sec16A and p115 are required for the proper positioning of TRAF3 along the mitochondrial network.

<p>(<b>A</b>) Confocal microscopy of HeLa cells transfected with 40 nM nonsilencing RNA duplexes (panels 1 and 2) or 40 nM siRNA duplexes that specifically target Sec16A (panels 3 and 4) or p115 (panels 5 and 6) and stained for MAVS and endogenous TRAF3 upon no treatment (panels 1, 3 and 5) or SeV infection (200 HAU/ml) for 4 h (panels 2, 4 and 6). Arrows indicate the colocalization of TRAF3 with MAVS. Bars represent 5 µm. One of three independent experiments with similar results is shown. (<b>B</b>) p115 and Sec16A were silenced in HeLa cells as described in (<b>A</b>) and infected with SeV for indicated periods of time. Whole-cell lysates were subjected to immunoprecipitation using an anti-TRAF3 (H-20) antibody followed by immunoblotting for the presence of MAVS and TRAF3. Immunoblot analysis against p115, Sec16A, TRAF3 and SeV proteins are also shown (Input). One of two independent experiments with similar results is shown. (<b>C</b>) Densitometric analysis of the binding activity of MAVS to TRAF3 presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002747#ppat-1002747-g006" target="_blank">Figures 6B</a>. Data represent the ratio of immunoprecipitated MAVS over immunoprecipitated TRAF3 and are means +/− S.D. of two experiments.</p

Activation of intracellular RNA and DNA sensors leads to the formation of TRAF3-contraining Golgi fragments.

<p>Confocal microscopy of HeLa cells stained for endogenous TRAF3, GM130, and the nucleus (DAPI) upon no treatment (<b>A</b>, panel 1), poly I:C treatment (<b>A</b>, panel 2) or poly dA:dT for 4 h (<b>A</b>, panel 3), infection with SeV (200 HAU/ml) (<b>B</b>, panel 1), RSV (MOI = 3) (<b>B</b>, panel 2), or Influenza A virus for 4 h (<b>B</b>, panel 3). Arrows indicate the relocalization and the colocalization of the Golgi apparatus with TRAF3 upon treatment. Bars represent 5 µm. One of three independent experiments with similar results is shown.</p

Through its ability to interact and colocalize with components of the ERES (Sec16A, depicted as thick green lines), ERGIC (ERGIC53 and p115) and the cis-Golgi apparatus (p115 and GM130), a subpopulation of TRAF3 (red circles) resides in the ER-to-Golgi vesicular compartment in non-infected cells.

<p>Upon dsRNA and dsDNA sensing, the cis-Golgi disorganizes into punctate structures, giving rise to membrane-bound compartments composed of at least GM130 and TRAF3 (dashed line). We propose that these membrane-bound compartments allow the proper positioning of TRAF3 with MAVS at Mitochondrial-Associated endoplasmic reticulum Membranes (MAM) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002747#ppat.1002747-Horner1" target="_blank">[86]</a>. There, being in close proximity with a component of the exocyst (sec5) and the translocon (Sec61β), TRAF3 allows the activation of TBK1 and IRF3 leading to activation of the type I IFN response. A similar scenario was recently proposed for STING (yellow circles) where in response to DNA virus infection, it traffics from the ER to the cis-Golgi apparatus and finally to a distinct perinuclear region for the activation of TBK1 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002747#ppat.1002747-Ishikawa2" target="_blank">[61]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002747#ppat.1002747-Saitoh1" target="_blank">[62]</a>. MTOC: microtubule-organizing center.</p